1. Field of the Invention
This invention lies in the field of transfection, the process by which exogenous molecular species are inserted into membranous structures by rendering the membranes of such structures permeable on a transient basis while the structures are in contact with a liquid solution of the species, thereby allowing the species to pass through the membranes.
2. Description of the Prior Art
The transformation of cells and other membrane-encased structures by the insertion of exogenous species, including species that are hydrophilic or otherwise membrane-impermeant, is of use in certain biologic and biochemical procedures. The transformation is generically referred to as transfection, and obtaining high efficiency in the procedure is a persistent problem. High efficiency in transfection means a high proportion of successfully transformed structures with minimal loss of viability of the structures, or at least with maximal restoration of the structures' viability upon the completion of the procedure by the natural processes of the structures themselves. A common way of performing transfection is by electroporation, which is the use of an electric field as the source of energy for permeabilization of the membrane. Standard electroporation procedures are performed in bulk, i.e., on populations of cells suspended in a buffer solution in which the species to be inserted is dissolved. Bulk electroporation entails high voltages, however, and provides little control over the effectiveness of the process on individual cells due to the difficulty of achieving uniform exposure of all of the cells to the electric field. Overexposure can result in irreparable damage to the cells while underexposure will fail to produce pores in the cell membrane. The differences among cells in a single population are due in part to the cells themselves since the cells will vary in size and stage of growth, and some cells will be shielded from the electric field by other cells.
To address these problems, systems for single cell electroporation have been developed. A system for adherent cells, for example, using microelectrodes of carbon fiber that are placed 2-5 microns from the cells, has been reported by Lundqvist, J. A., et al., “Altering the biochemical state of individual cultured cells and organelles with ultramicroelectrodes,” Proc. Natl. Acad. Sci. U.S.A., 1998, 95, 10356-10360. A system for non-adherent cells using electrolyte-filled capillaries has been reported by Nolkrantz, K., et al., “Electroporation of Single Cells and Tissues with an Electrolyte-Filled Capillary,” Analytical Chemistry, 2001, 73, 4469-4477. Systems using micropipettes have been reported by Haas, K., et al., “Single-cell electroporation in gene transfer in vivo,” Neuron, 2001, 29, 583-591, and by Rae, J. L., et al., Eur. J Physiol., 2002, 443, 664-670. A system using a specially designed microelectroporation chip is described by Huang, Y., et al., “Micro-Electroporation: Improving the Efficiency and Understanding of Electrical Permeabilization of Cells,” Biomed. Microdevices, 1999, 2:2, 145-150. A system that draws a small section of the cell membrane into a channel and then applies an electric field between two electrodes, one inside and the other outside the channel, is reported by Ionescu-Zanetti, C., et al., United States Patent Application Publication No. US 2007/0243523 A1, “Methods and Apparatus for Manipulation of Particle Suspensions and Testing Thereof” (Oct. 18, 2007). Additional disclosures of single-cell electroporation of possible relevance to the present invention are those of Khine, M., et al, “Single Cell Electroporation Chip,” Lab Chip, 2005, 5, 58-43; Khine, M., et al., “Single-cell electroporation arrays with real-time monitoring and feedback control,” Lab Chip, 2007, 7, 457-462; and Nolkrantz, K. et al., “Functional Screening of Intracellular Proteins in Single Cells and in Patterned Cell Arrays Using Electroporation,” Analytical Chemistry, 2002, 74, 4300-4305.